The present invention relates to a method of generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest.
The advent of recombinant DNA techniques has made it possible to select single protein components with interesting properties and produce them on a large scale. This represents an improvement over the previously employed production process using microorganisms isolated from nature and producing a mixture of proteins which would either be used as such or separated after the production step. Methods have been developed for rapid identification of genes encoding a polypeptide of interest.
One example is the so called expression cloning technique described in WO 93/11249 (Novo Nordisk A/S). The technique disclosed in WO 93/11249 comprises a method of screening for a DNA sequence in a DNA library prepared from an organism suspected of producing genes encoding polypeptides with activities of interest. Such a library has traditionally been made on DNA isolated from a single known microorganism.
A compartmentalization method of screening microorganisms having a selectable characteristic has previously been devised in WO 97/37036, and a process for forming a normalized genomic DNA library from an environmental sample is described in WO 97/37036.
However, a method of generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest has never been described. Therefore, it would be desirable to have a method based on biological enrichment for selecting potentially interesting genes from environmental pool of organisms.
It has now been found possible to use biological enrichment for selecting potentially interesting genes from an environmental pool of organisms. Accordingly, the invention provides a method for generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or under conditions suitable for enriching said pool of organisms in organisms harbouring said DNA; and
b) preparing a gene library from the resulting enriched pool of organisms.
The invention also provides a method of selecting a DNA sequence of interest from an environmental pool of organisms, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or conditions suitable for enriching said pool of organisms in organisms harbouring said DNA sequence;
b) producing gene libraries from the resulting enriched pool of organisms;
c) screening the libraries for DNA containing the desired gene; and
d) selecting the DNA sequence of interest resulting from the screening of step c).
Further, the invention relates to a gene library prepared from an enriched environmental pool of organisms enriched in DNA encoding a polypeptide with an activity of interest.
It is an object of the present invention to provide a method for generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or under conditions suitable for enriching said pool of organisms in organisms harbouring said DNA; and
b) preparing a gene library from the resulting enriched pool of organisms.
In the context of the present invention, the term xe2x80x9can environmental pool of organismsxe2x80x9d means a environmental sample comprising microorganisms and cells from higher animals harboring DNA encoding a polypeptide with an activity of interest. The environmental sample may for instance be an environmental sample of soil or plant material, animal or insect dung, insect gut, animal stomach, a marine sample of sea or lake water, sewage, waste water, a sample of sludge or sediment, etc., comprising one or, as in most case, a vast number of different microorganisms or living cells.
In step a), the sample as such is cultivated without any need for further purification. By selecting the medium and the cultivation conditions at which the sample is cultivated, it is possible for enriching or (amplifying) organisms having optimal growth at the specific cultivation conditions, and expressing polypeptides with properties adapted to the cultivation conditions. The gene library prepared in step b) may be prepared by any suitable technique known in the art, non-limiting examples of which are described in Example 3 and 4.
The advantage presented by the present screening method is primarily that the rate at which novel genes may be isolated and, consequently, novel products be developed may be greatly increased. Furthermore, the method permits screening for multiple polypeptides activities and may even result in the isolation of several different genes coding for the same type of polypeptides.
By use of the invention it is possible to exploit enriched cultures for detecting novel enzymes, and other polypeptides with an activity of interest.
In a preferred embodiment, the method of the invention comprises subjection the environmental pool of organisms to cultivation in a medium, which contains a substrate for the polypeptide with the desired activity. A wide range of substrates for the enrichment of the environmental of organisms containing different types of gene products may be used. For instance, a DNA encoding a polypeptide with an activity of interest such as a pectinase enzyme may be selected as a gene product on a substrate as pectin.
In a preferred embodiment, the substrate constitutes the carbon source and/or nitrogen source of the medium.
In a more preferred embodiment, the substrate comprises pectin, amylose, cellulose, galactan, xylan, arabinan, mannan, lipid or hemicellulose or a combination thereof.
In a preferred embodiment of the method of the invention, the enrichment is achieved by one or more growth conditions. In a another preferred embodiment, the growth conditions comprise pH and temperature. In yet another preferred embodiment, the growth conditions of step a) used for achieving the enrichment comprises any pH range ie. 0-12, preferably of about 6-9, in particular 9-12, at any temperature range i.e. 0-120xc2x0 C., preferably about 25-30xc2x0, preferably 30-500xc2x0, most preferred 50-70xc2x0 C.
An important step in the procedure for selection of a potentially interesting environmental pool of organisms is to select the optimal pool to start from. To select genes encoding polypeptides that can break down natural compounds of plant (or animal) origin, it is preferable to look into those biotopes in nature where such materials are efficiently decomposed. Examples of animals especially efficient in breaking down plant material are the ruminates, termites and insects (sensu lato).
In a preferred embodiment, the environmental pool of microorganisms is isolated from an animal stomach or an insect gut.
In a more preferred embodiment, the pool of microorganisms is isolated from a cow""s rumen.
Likewise, it is important when selecting genes encoding polypeptides with an activity of interest that are capable of working under e.g. strongly alkaline conditions, to isolate the pool of organisms from an equally strongly alkaline biotope. It is known in the art that in order for Bacillus thuringensis (Bt) toxins to be active, strongly alkaline conditions are a prerequisite [Bacillus thuringensis, an environmental biopesticide: Theory and Practice, 1993, eds. P. F. Entwistl et al., Wiley and Son, UK]. The guts of the larvae belonging to the orders of insects known to be sensitive to the Bt toxins comprise environmental pools of a high alkaline nature (approx pH 10). Such insect orders are especially Isoptera, Lepidoptera, Coleoptera and Diptera.
Consequently in a preferred embodiment, the pool of microorganisms is isolated from the gut of an insect of the Isoptera, Lepidoptera, Coleoptera, or Diptera families.
In a more preferred embodiment, the pool of microorganisms is isolated from the gut of insects selected from the group consisting of Agrotis, Neotermes castaneus, Tineola bisselliella, and Melolontha vulgaris. 
Prior to isolating environmental pools of organisms from animal stomachs or insect guts, it is interesting to do an enrichment by rearing or supplying the animal or insect with food comprising a substrate for the activity of the polypeptide of interest, maybe even as the primary carbon and/or nitrogen source. This makes a cow""s rumen and larval guts from Lepidopteran, Coleopteran and Dipteran species highly interesting for further enrichment through feeding with specific substrates.
In a preferred embodiment the pool of microorganisms is enriched by supplying feed to the animal or insect, which comprises a substrate for the polypeptide with an activity of interest.
Specific examples of xe2x80x9cDNA encoding a polypeptide with an activity of interestxe2x80x9d include among others enzymatic activity and anti-microbial activity.
In a preferred embodiment, the gene libraries are enriched in DNA encoding an enzyme activity of interest.
In a more preferred embodiment of the invention the activity of interest is an enzymatic activity, such as an activity selected from the group comprising of phosphatases oxidoreductases (E.C. 1), transferases (E.C. 2); hydrolases (E.C. 3), such as esterases (E.C. 3.1), in particular lipases and phytase; such as glucosidases (E.C. 3.2), in particular xylanase, cellulases, hemicellulases, and amylase, such as peptidases (E.C. 3.4), in particular proteases; lyases (E.C. 4); isomerases (E.C. 5); ligases (E.C. 6).
In another preferred embodiment, the enzyme of interest comprises a protease, lipase, beta-galactosidase, lactase, polygalacturonase, beta-glucoamylase, esterase, hemicellulase, peroxidasee, oxidase, laccase or glucose oxidase.
In more preferred embodiment, the enzymes obtained in the method is an amylase, in particular an xcex1-amylase or a xcex2-amylase, an arabinanase, an arabinofuranosidase, a galactanase, an xcex1-galactosidase, a xcex2-galactosidase, a polygalacturonase, a pectin methyl esterase, a rhamnogalacturonase, a rhamnogalacturon acetyl esterase, a pectin lyase, a xylanase, a cellulase, a xcex2-glucosidase, a cellobiohydrolase, a xylosidase, a mannanase, and/or a glucuronisidase.
The environmental pool of organisms containing DNA encoding a polypeptide with an activity of interest are typically microorganisms such as Eubacteria, Archaebacteria, fungi, algae and/or protozoa.
The polypeptide may be an enzyme of interest obtained from any known organism. Preferably the enzyme may be obtained from microorganism, in particular from a bacteria, from a filamentous fungus or a yeast.
In the method of the invention, the organisms are enriched cultures meaning that the cultures are selected on a specific substrate from which other organisms are not able to grow or having a reduced growth.
It is another object of the invention to provide a method of selecting a DNA sequence of interest from an environmental pool of organisms, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or conditions suitable for enriching said pool of organisms in organisms harbouring said DNA sequence;
b) producing gene libraries from the resulting enriched pool of organisms;
c) screening the libraries for DNA containing the desired gene; and
d) selecting the DNA sequence of interest resulting from the screening of step c).
Step a) and b) are as described above. In step c), clones found to comprise a DNA sequence originated from the prepared gene library in step b) may be screened for any activity of interest. Examples of such activities include enzymatic activity, anti-microbial activity or biological activities. In step c), gene libraries are screened for genomic DNA containing the desired gene, and in step d), the DNA sequence of interest are selected from the screening of step c). Step c) and d) may be performed by standard methods known in the art.
The polypeptide with the activity of interest may then be tested for a desired performance under specific conditions and/or in combination with e.g. chemical compounds or agents. The gene libraries may be screened according to the method of the invention for a polypeptide with an activity of interest e.g. a specific activity, and/or a specific property of interest such as thermostability, high pH tolerance, wash performance, textile dyeing, hair dyeing or bleaching properties, a effect in feed or food ect. The appropriate assay for testing for a desired activity and/or property well be known to the skilled person.
In a preferred embodiment of the method, the gene library comprises an enzyme-encoding gene of interest, and the gene library is screened for enzymes under conditions which the enzyme is active. This means that the library may be screened for enzymes at e.g. high ,temperatures such as 60-110xc2x0 C. and high pH such as 10-12 e.g. in cases where it is desired to isolate a DNA sequence encoding an alkaline enzyme with a relatively high thermostability. However, pH can be in any range e.g. of from about 0 to about 12, and the temperature in any range e.g. of from about 5 to about 110xc2x0 C., preferably of from about 60 to about 90xc2x0 C.
It is still another object of the invention to provide a gene library prepared from an environmental pool of organisms enriched in DNA encoding an polypeptide with an activity of interest. In a preferred embodiment, the gene library comprises a polypeptide with an activity of an enzyme, a hormone or a toxin. In a more preferred embodiment, the gene library comprises an enzyme activity of interest as described above.